Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylose is an unbranched chain of up to several thousand α-D-glucopyranose units linked by α-(1,4)-glycosidic bonds. Amylopectin is a highly branched molecule made up of up to 50,000 α-D-glucopyranose residues linked by α-(1,4) and α-(1,6) glycosidic bonds. Approximately 5% of the glycosidic linkages in amylopectin are α-(1,6) bonds, which leads to the branched structure of the polymer.
Amylose and amylopectin molecules are organized into starch granules that are stored in plastids. The starch granules produced by most plants are 15 to 30% amylose and 70 to 85% amylopectin. The ratio of amylose to amylopectin and the degree of branching of amylopectin affect the physical and functional properties of the starch. The usefulness and value of starches in food and industrial applications is determined by functional properties such as viscosity and stability. Specific functional properties may be obtained by using the starch from a crop such as corn, rice, potatoes, or wheat which meets said functional properties. If no starch is found which meets the required functional property, such as the need for stable viscosity under high temperatures and acidic conditions, the functionality can usually be achieved by chemically modifying the starch. Various types and degrees of chemical modification are used in the starch industry, and the labeling and use of chemically modified starches must meet government regulations.
Within the starch bearing organs of plants, the proportion of amylose to amylopectin and the degree of branching of amylopectin are under genetic control. For example, corn plants homozygous for the recessive waxy (wx) mutation lack a granule-bound starch synthase enzyme and produce nearly 100% amylopectin. Corn plants homozygous for the recessive amylose extender (ae) mutation and uncharacterized modifier genes can reportedly produce starch granules that are approximately 80 to 90% amylose (see U.S. Pat. No. 5,300,145). The dull mutant of corn lacks a starch synthase distinct from the one lacking in the waxy lines. The starch from the dull mutant is characterized by more amylose and a larger proportion of shorter branches on the amylopectin molecule than normal starch.
Most cereal crops are handled as commodities, and many of the industrial and animal feed requirements for these crops can be met by widely grown and volume-produced common varieties. However, at present, there exists a growing market for crops with special end-use properties not met by grain of standard composition. Most commonly, specialty corn is differentiated from “normal” corn by altered endosperm properties. For example, waxy or high amylose corn contains an overall change in the ratio of amylose to amylopectin, sweet corn contains an increased accumulation of sugars, and food-grade corn and popcorn contain an alteration in the degree of endosperm hardness (Glover, D. V. and Mertz, E. T. (1987) in Corn: Nutritional Quality of Cereal Grains; Genetic and Agronomic Improvement, R. A. Olson and K. J. Frey, eds. American Society of Agronomy, Madison Wis., pp. 183-336; Rooney, L. W. and S. O. Serna-Saldivar, (1987) Food Uses of Whole Corn and Dry-milled Fractions, in Corn: Chemistry and Technology, S. A. Watson and P. E. Ramstead, eds. American Association of Cereal Chemists, Inc., St. Paul, Minn., pp. 399-429). The present invention offers the buyers of specialty grains a source of starch having properties distinct from starch derived from commodity crops, also known as dent starch, and offers farmers the opportunity to grow a higher value-added crop than conventional or commodity corn.
Purified starch is obtained from plants by a milling process. Starch is extracted from corn kernels through the use of a wet milling process. Wet milling is a multi-step process involving steeping and grinding of the kernels and separation of the starch, protein, oil and fiber fractions. A review of the corn wet milling process is given by S. R. Eckhoff (1992) in the Proceedings of the Fourth Corn Utilization Conference, June 24-26, St. Louis, Mo., printed by the National Corn Growers Association, CIBA-GEIGY Seed Division and the United States Department of Agriculture. Wheat is also an important source of purified starch. Wheat starch production is reviewed by J. W. Knight and R. M. Olson (1984) in Starch: Chemistry and Technology 2nd Edition, Academic Press Whisler et al. Eds.
Starch is used in numerous food and industrial applications and is the major source of carbohydrates in the human diet. Typically, starch is mixed with water and cooked to form a thickened gel. This process is termed gelatinization. Three important properties of a starch are the temperature at which gelatinization occurs, the viscosity the gel reaches, and the stability of the gel viscosity over time. Distinct differences in gelatinization behavior can be found for starches from different crops and for maize starches of different genotypes. These differences may be attributed to variations in the amylose to amylopectin ratio, the composition of the starch and the amylopectin branch-chain distribution. As the temperature of a solution of dent starch in water is increased, viscosity will increase as the starch granules swell and take up water. A maximum viscosity will be attained before the granules rupture and the granule contents are released into solution. With cooling, amylose chains reassociate to form a more organized structure and viscosity will once again increase as a stiff gel is formed. Waxy starches that lack amylose cook at a lower temperature and with cooling tend to form softer gels. However, the amylopectin branch chain distribution of particular waxy starches can also significantly influence gelatinization temperature, viscosity increase and the propensity of a cooked starch to form a stiffened gel (Jane, J. et al (1999) Cereal Chem. 76:629-637).
The amylose content in cornstarch affects many physical and functional properties of starch including crystalline structure, pasting temperature, gel formation, and resistance to digestion. In general, as amylose levels are increased, crystallinity as measured by x-ray diffraction or birefringence is decreased, heat capacity and pasting temperature are increased, and stiffer gels are produced (Cheetham, N. W. H. and Leping, T. (1998) Carbohydr. Polym. 36:227-228; Kalistratova, E. N. (1999) Starch/Starke 51:160-162; Jane, J. et al. (1999) Cereal Chem. 76:629-637). This influence of amylose on starch functional properties is such that, for certain applications, waxy starches, containing no amylose, are preferred. However, the attributes conferred by amylose-containing starches are desirable in certain recognized applications and it is conceivable that additional utility may be further demonstrated. High amylose starches are sources of resistant starch that serve as a dietary fiber. These starches are also of use in food coatings, starch jelly confections, films, and biodegradable plastics (Campbell, M. R. et al. (1999) Cereal Chem. 76:552-557).
Synthesis of the two starch polymers and their assembly into the starch granules is an area of intense research. Biosynthesis of amylopectin involves the participation of starch synthases, starch branching enzymes, and starch debranching enzymes via a complex process that, to this day, remains poorly understood. While the roles that these enzymes play in the synthesis of the branched polymer are not completely defined, the critical involvement of a specific starch synthase, granule-bound starch synthase I (GBSSI) in the synthesis of amylose in starch storing tissues has been demonstrated. Mutants lacking this enzyme activity have been identified in corn, potato, rice, and pea (Shure, M. et al. (1983) Cell 35:225-233; Hovenkamp-Hermelink, J. H. M. et al. (1987) Theor. Appl. Genet. 75:217-221; Sano, Y. (1987) Theor. Appl. Genet. 68:467-473; and Denyer, K. et al. (1995) Plant Cell Environ. 18:1019-1026). Starch isolated from these mutants contains little, if any, of the linear starch polymer, amylose. Introduction of a wild type potato GBSSI gene into amf potato by Agrobacterium-mediated transformation leads to complementation of the genetic defect in this mutant. Granule bound starch synthase activity is restored and amylose levels reach amounts similar to those found in “starch cultivars” of potato (van der Leij, F. R et al. (1991) Theor. Appl. Genet. 82:289-295).
Amylose amounts vary with the plant source but generally fall within the range of 15 to 30% of the total starch content. It has been suggested that the amount of amylose that normally accumulates in wild type plants may be determined by one or more factors functioning separately or jointly. These factors may be physical constraints, substrate supply (i.e. ADP-glucose), level of GBSSI activity, or availability of oligosaccharide primers (Smith, A. M. et al. (1997) Annu. Rev. Plant Mol. Biol. 48:67-87). Amylose chain length is also found to vary depending upon the botanical source of the starch. An evaluation of amylose samples from 7 different species has indicated that small amylose, with a degree of polymerization (dp) lower than 1000, is predominant in cereals while that with a dp larger than 1000, is found in tuberous plants (Hanashiro, I. and Takeda, Y. (1998) Carbohydr. Res. 306:421-426). The degree of polymerization of maize amylose has been reported to be 800 while that of potato is 3000 (Ellis, R. P. et al. (1998) J. Sci. Food Agric. 77:289-311). The factors that are responsible for determining amylose level and degree of polymerization in higher plants have not been systematically studied and are not currently known.
In tubers, the ability of a heterologous GBSSI from cassava to complement the amf mutation in potato was recently reported. In this study, Agrobacterium-mediated transformation was used to introduce into the amf mutant either a complete copy of the cassava (Manihot esculentum) GBSSI coding region, or hybrid versions containing sequences from both the potato and the cassava proteins. One of the hybrid proteins consisted of the potato GBSSI transit peptide fused to the remainder of the mature cassava protein. Another comprised the potato GBSSI transit peptide, the first 89 amino acids of the mature potato GBSSI (containing the substrate-binding site for ADP-glucose) protein followed by the mature cassava GBSSI protein Expression of the native cassava protein gave only partial complementation with amylose levels reaching 8.2% of the total starch. This restores amylose to 37% of the wild type level. Expression of these hybrid proteins gave the best results. The best performing plants accumulated 13% amylose compared to 22% amylose present in the wild type. Only transformants containing the hybrid protein consisting of the transit peptide and the first 89 amino acids of potato GBSSI fused to the remainder of the cassava GBSSI protein accumulated amylose in excess of 10% of the total starch. These levels of amylose polymer were found in only 13% of the transformants expressing this hybrid protein. Physicochemical determinations performed on the transgenic starch confirmed the presence of amylose in the complemented lines. Measured parameters obtained from Bohlin rheometry and differential scanning calorimetry were intermediate between those displayed by wild type and amf starch and were related to the amount of amylose present in the investigated transgenic lines. No measurement of the amylose degree of polymerization was reported. (Salehuzzaman, S. N. I. M. et al. (1999) Plant Cell Environ. 22:1311-1318).
Salehuzzaman et al. contend that the failure to completely restore amylose to wild type levels via introduction of a heterologous cassava GBSSI or a potato-cassava hybrid GBSSI protein can be explained by inherent differences in the intrinsic properties of the potato and cassava proteins. Indeed, amylose amounts of 10% or more of the total starch are reached only upon expression of the hybrid protein containing the substrate-binding site for ADP-glucose that is derived from the potato GBSSI protein.
Identification of the factors involved in determining amylose level and degree of polymerization in higher plants will permit the generation of transgenic plants where the levels of amylose and its fine characteristics may be manipulated.